Layered expression scanning (LES), or multimembrane blotting, is a recently developed method for the high-throughput analysis of proteomic profiles from multiple samples of physiological fluids or tissue sections (See C. R. Englert, et al, “Layered expression scanning; Rapid molecular profiling of tumor samples,” Cancer Res., 60 (2000) 1526; A. L. Feldman et al., “Modulation of tumor-host interactions, angiogenesis, and tumor growth by tissue inhibitor of metalloproteinase 2 via a novel mechanism,” Cancer Res., 64 (2004) 4481, incorporated herein by reference). If applied to a 2-D gel, in which proteins have been separated by molecular weight in one dimension and by charge in the other, LES provides a third dimension related to protein function.
As originally developed, a layered stack of membranes with straight-through pores is used, onto which proteins bind nonspecifically to each membrane during sample transfer. After transfer through the stack, each membrane is exposed to a different antibody that binds to a particular protein of interest. Low porosity membranes with straight-through pores are used, rather than the highly-porous nitrocellulose or PVDF membranes with interconnected pores traditionally used in blotting, to prevent image distortion due to lateral diffusion, thus producing “carbon copies” of each sample from a single transfer. Minimal lateral diffusion is particularly important for maintaining morphological features in tissue sections.
However, as a result of nonspecific binding (universal capture), proteins are depleted as they pass through the membrane stack. This can significantly reduce the sensitivity of the multimembrane approach and, as a result, only a limited number of proteins can be analyzed. Therefore, there is significant motivation to develop affinity or “smart” membranes, each of which can selectively capture one specific protein. A highly selective affinity membrane will drastically reduce protein depletion and increase the sensitivity of the LES technique. Such a membrane will also be advantageous in conventional blotting if the target protein is present at very low concentrations.
The development of an affinity membrane for blotting applications requires the immobilization of capture biomolecules (antibodies, proteins, polypeptides) on the surface of a base membrane. The immobilization of an antibody (IgG) to a solid surface is currently used in a number of diagnostic assays, such as the enzyme-linked immunosorbent assay (ELISA) and antibody arrays. The primary challenges for creating any surface with immobilized proteins are to prevent denaturing during immobilization and to orient the proteins to maintain bioactivity.
A method and composition for the identification of a target biomolecule in a sample has been developed. The method comprises obtaining a stack of coated capture membranes comprising a plurality of capture membranes, each coated with a different capture biomolecule. The membrane stack is exposed to a sample, and, after a given amount of time for the sample to permeate the membrane stack, the membrane stack is removed from the sample carrier and the capture membrane to which the target biomolecule adheres is identified. However, there are differences between antibodies and other proteins or peptides that require different techniques for attachment to a membrane.
For antibodies, attachment entails immobilizing the Fc tail such that the Fab fragments, where specific target binding occurs, are accessible and properly oriented. Several different methods have been reported in the literature for surface immobilization of antibodies. These techniques include: (1) direct spotting of antibody on a solid surface (Knezevic, V, Proteomic profiling of the cancer microenvironment by antibody arrays,” Proteomics, 1 (2001) 1271, incorporated herein by reference); covalent attachment of antibody on a chemically activated surface using glutaraldehyde chemistry or through a variety of other chemistries, (Angenendt, et al., “Toward optimized antibody microarrays: a comparison of current microarray support materials,”Anal. Biochem., 309 (2002) 253; W. Kusnezow, et al, “Antibody microarrays: An evaluation of production parameters, ” Proteomics, 3 (2003) 254; and U. B. Nielsen et al.“Multiplexed sandwich assays in microarray format,” J. Immunol. Methods, 290 (2004) 107 (herein incorporated by reference); an (3) immobilization via affinity tags (H. Zhu et al., “Global analysis of protein activities using proteome chips,” Science, 293 (2991) 2101 and Johnson, C. P. et al, “Engineered protein A for the orientational control of immobilized proteins,” Bioconjugate Chem., 14 (2003) 974 incorporated herein by reference); (4) biotinylation of capture molecules and their immobilization on streptavidin coated support (Delehanty, J. B. et al, “A microarray immunoassay for simultaneous detection of proteins and bacteria,” Anal. Chem., 74 (2002) 5681 and Peluso, P. et al. “Optimizing antibody immobilization strategies for the construction of protein microarrays,”Anal. Biochem., 312 (2003) 113, herein incorporated by reference); (5) immobilization of IgG on Protein A or Protein G coated surfaces (Kumar, A. et al. “Emerging technologies in yeast genomics,” Nat. Rev. Genet., 2 (2001) 302 and Vijayendran, R. A. and Leckband, D. E., “A quantitative assessment of heterogeneity for surface-immobilized proteins,”Anal. Chem., 73 (2001) 471, incorporated herein by reference); (6) DNA-directed immobilization (DDI) for proper orientation of antibodies (Wacker, R. et al. Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study,” Anal. Biochem., 330 (2004) 281, herein incorporated by reference); and (7) cutinase- or AGT-fusions (Kwon, Y. et al. “Antibody arrays prepared by cutinase-mediated immobilization on self-assembled monolayers,” Anal. Chem., 76 (2004) 5713 and Sielaff, I. et al. “Protein function microarrays based on self-immobilizing and self-labeling fusion proteins,” Chem Bio Chem, 7 (2006) 194, herein incorporated by reference).
One of the most commonly used methods to immobilize proteins is the nonspecific reaction of amine groups, both on the surface and in the protein, with glutaraldehyde Given the large number of amino acid residues with pendant amine groups in a typical protein, this approach often crosslinks the protein, leading to significant denaturing and a small fraction of active protein. For example, amine functional surfaces have been reacted with Protein A or Protein G to take advantage of the ability of these proteins to bind to the Fc tail on IgG as a means of orienting antibodies on the surface. Although the use of Protein A (or G) improves the activity of antibody relative to glutaraldehyde immobilization of the antibody itself, a significant fraction of the Protein A (or G) is denatured during surface immobilization and cannot orient antibodies properly upon binding.
One method for improving the number of active biomolecules on a surface is to use specific biological interactions to link the biomolecule to the surface in an effort to control its orientation. An example for the use of such interactions is immobilized metal affinity chromatography (IMAC), which is based on the chelation of specific amino acids, such as histidine, to cations, such as Ni++, that are bound to groups in the polymeric media. Histidine tags are often engineered onto the terminal ends of proteins specifically for separation on IMAC columns. Recently, it was reported that the immobilization of hexahistidine-tagged Protein A (his-Protein A) on Ni++-nitrilotriacetic acid (NTA) and Ni++-hydrosuccinimide derivative surfaces demonstrated that his-Protein A can properly orient IgG. (See Johnson, infra).